This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Objective: To establish system for efficient differentiation of hESC into RBCs with potential to use for blood transfusions. To generate erythroid cells from hESCs, as a first step, we cocultured hES cells with OP9 bone marrow stromal cell line for 6 days to induce their differentiation towards CD34+ cells, which included population of CD34+CD43+ hematopoietic progenitors (10-20%), CD34+CD43-KDR+ endothelial cells (up to 60%), and CD34+CD43-KDR- mesenchymal cells (less than 15%). Subsequently CD34+ cells were isolated using magnetic sorting and cultured in non-adherent conditions in SFEM supplemented with SCF, TPO, EPO, IL-3, IL-6, EX-CYTE, insulin, dexamethasone, and transferrin for five days. From the day 6, cells were expanded in the same media without TPO, IL-3 and IL-6. After 10 days of culture, most of the cells were immature CD71+CD235a+ erythroid progenitors expressing high level of embryonic [unreadable]- and fetal [unreadable]-globin, and low level of adult [unreadable]-globin as determined by PCR. Erythroid progenitors continued proliferate in culture for up to 60 days resulting in more than 4000-fold expansion. Following expansion, [unreadable]-globin expression increased and [unreadable]-globin expression decreased and eventually disappeared by the day 50 of culture. Morphologic evaluation of expanded cells revealed homogenous population of erythroid progenitors at different stages of maturation, including erythroblasts and normoblasts. By flow cytometry, essentially all cells were CD71+CD235a+ and CD34- and CD45-, confirming that they represent pure population of erythroid progenitors free of leukocytes. The described experimental system will be useful for the studies of genes regulating erythroid cell expansion, as well as molecular mechanisms regulating globin gene expression and switches during early hematopoietic development as well for large scale production of red blood cells from hESCs for clinical purposes. This research used WNPRC Stem Cell Resources. This research used start-up funds;there are no publications yet.